RECENT FINDINGS: STRUCTURAL AND FUNCTIONAL STUDIES OF ORF1p - ORF1p is one of two L1 encoded proteins. Earlier studies by others of mouse ORF1p showed that it binds nucleic acids, acts as a nucleic acid chaperone, and forms trimers via a highly conserved coiled coil domain. However, the function of ORF1p in retrotransposition is largely unknown. We are using several approaches to examine this problem including analysis of the structural, biochemical, and biological effects of positive selection, which involved mainly the coiled coil domain (Boissinot, et al, Mol. Biol. Evol. 18: 2186). To do so we resuscitated an ORF1p from an extinct L1 family, L1Pa5, which is ancestral to the modern human(h) ORF1p of the currently active human L1Pa1 family. We also created mosaic versions of ORF1p that contain modern and ancestral regions, and other variants that were deleted of various domains. We extensively characterized these proteins with respect to retrotransposition and interaction with several mammalian host protein in vivo. In addition, we purified mg amounts of some the various of ORF1ps to homogeneity. We examined their various biophysical and biochemical properties in vitro including several assays that reflect their nucleic acid chaperone activity. We uncovered two novel properties of ORF1p in 2012: That hORF1p trimers can reversibly polymerize under the conditions required for high affinity nucleic acid binding, and that this property is involved in the second novel property of the protein, namely its biphasic effect of mismatched double-stranded nucleic acids, protecting it from dissociation (melting) at low concentrations, but melting it at high concentrations largely polymeric. A mismatched duplex is a proxy nucleic acid chaperone substrate. Thus, determining the biophysical basis the surprising and novel diphasic affect of the protein on this substrate is essential to our further progress in this area. Thus, in early 2013 we began a collaboration with Dr. Mark Williams to study these interactions by atomic force microscopy. This year, also implemented a new sensitive FRET assay for assessing nucleic acid chaperone activity under equilibrium conditions, carried out extensive mutational analysis of coiled coil domain in an attempt to clarify the biochemical effect of positive selection in this region of the protein, and determined that ORF1p is phosphorylated at a number of sites which we subjected to mutational analysis. Over the past year we made significant progress on both projects: With regard to the coiled coil we found that change of any one of just 4 amino acids of a modern adapted coiled coil to their ancestral counterparts eliminate retrotransposition and that activity is only restored by converting 17 additional modern residues in the coiled coil to their ancestral counterparts to restore activity. That concerted changes are required to compensate for the effects of single amino acid changes provides a rationale for positive selection during evolution of the coiled coil and indicates that cross talk between different regions of the coiled coil can extend over long distance and is crucial for maintaing a functional structure. Determining the basis of this cross talk could offer major insights as to how the trimeric structure is related to its role in retrotransposition. With regard to the phosphorylation studies we just submitted a paper to PNAS which reports that L1 replication depends on phosphorylation ofORF1p. Thus L1 is integrated with, and perhaps competes for and perturbs the host regulatory systems responsible for numerous essential host processes such as cell division and differentiation. These findings represent not only a major advance in our understanding of L1 regulation but also suggest that the effect of L1 on its host is likely far more profound than just an agent of genetic change.